Proposal Summary/Abstract Disordered regulation of hepatic lipid metabolism is found in a variety of important disorders. In particular, nonalcoholic fatty liver disease (NAFLD) is a disorder of hepatic lipid homeostasis in which both cholesterol and triglycerides accumulate in hepatocytes. A fraction of patients with NAFLD progress to liver cell necrosis, inflammation, and progressive fibrosis. NAFLD is a now recognized as a leading cause of cirrhosis in the U.S. Hepatic lipid accumulation in NAFLD appears to produce injury in part by inducing the unfolded protein response (UPR) in the endoplasmic reticulum; a progression of similar events to those found with the development of atherosclerosis. As with atherogenesis, treatments that reduce intracellular lipid accumulation may attenuate liver injury in NAFLD by repressing the UPR. Recently, we have identified mitochondrial cholesterol delivery and oxidation as crucial steps in the regulation of hepatic lipid metabolism. Increased expression of the mitochondrial cholesterol delivery protein, StARD1, in hepatocytes was found to down-regulate pathways of cholesterol biosynthesis while up-regulating pathways of cholesterol degradation and secretion. Consequences of StARD1 overexpression include markedly decreased intracellular neutral lipids (cholesterol and triglycerides), increased levels of key nuclear receptors important in lipid homeostasis, and reduced expression of HMG CoA reductase, acetyl CoA carboxylase, and fatty acid synthase (rate-determining enzymes in the biosynthesis of cholesterol and fatty acids). We now have shown that hepatic StARD1 overexpression increases cholesterol oxidation via pathways initiated by mitochondrial CYP27A1, and the resulting oxysterol products are regulatory molecules capable of mediating the resulting changes in lipid metabolism. CYP27A1 is a ubiquitous mitochondrial enzyme, and our preliminary data indicate that the StARD1/CYP27A1 pathway may regulate lipid homeostasis in many extrahepatic tissues as well. In the hereditary disorder CTX, caused by genetic deletion of CYP27A1, absence of these regulatory oxysterols is associated with accumulation of lipids in various tissues (inclusive of the liver), accelerated atherosclerosis, and neurologic impairment. The objective of this renewal application is to further elucidate the role of the StARD1/CYP27A1 pathway of cholesterol oxidation in the regulation of hepatic lipid homeostasis. We hypothesize that StARD1 serves as an intracellular sensor of cholesterol availability. When cholesterol is present in excess, mitochondrial cholesterol delivery increases, leading to increased production of CYP27A1 derived oxysterols. The resulting oxysterols then modulate lipid metabolism by binding to nuclear receptors. We further hypothesize that stimulation of this pathway in the liver could represent a useful strategy for treatment of nonalcoholic fatty liver disease. Four specific aims are proposed to study this hypothesis. Specific aim 1 will use selective StARD1 overexpression in intact and knock-out models to determine if StARD1/CYP27A1 pathway derived oxysterols are responsible for activating key nuclear receptors that control the expression of genes involved in the regulation of cholesterol, fatty acid, and bile acid homeostasis; and, subsequently determining how expression of the encoded pathways correlates with respective nuclear receptor activation. In Specific aim 2, we propose to characterize and assess mechanisms of activation of nuclear receptors by StARD1/CYP27A1 pathway derived oxysterols involved in lipid homeostasis. Specific aim 3 will determine the role of the StarD1/CYP27A1 pathway of cholesterol metabolism in attenuating the unfolded protein response (UPR) in hepatocytes. Specific aim 4 will test for the first time the pharmacologic potential of increased StARD1 expression to prevent or reverse disorders of liver lipid accumulation in animal models representative of human disease.